Method for obtaining novel derivatives of naphthalene for the in vivo diagnosis of alzheimer&#39;s disease

ABSTRACT

This invention relates to a chemistry branch, particularly to the field of compounds&#39; organic synthesis that belongs to the aromatic bicyclic or naphthalene category, used in the detection of amyloid sheets. These new naphthalene derivatives have a general formula: Wherein R represents mutually independent groups. In I: R 1 :-alkylenyl-C(O)NH-alkylenyl-R 3 , -alkylenyl-C(O)O—R 4 , R 3 :—COOH, —OH, —SH, —NH 2 , -alkyl-NH-alkyl-N-dithiocarbamate alkaline earth metal salts, R 4 : H, succinimidyl group, R 2 : —H,-alkyl. In II: R 1 : -alkyl, -alkylenyl-halide-alkylenyl-hydroxyl-alkylenyl-O-aryl, —O-alkylsulfonate alkylenyl, R 2 : -halide-alkylenyl-O-aryl, -alkylenyl-O-alkylsulfonate, -alkylenyl-halide-, —CH(O), —HC═C(CN) 2 , —HC═CHNO 2 , -alkylenyl-NH 2 , -alkylenyl-NH-alkyl, -alkylenyl-alkyl-N-dithiocarbamate alkaline salts. The terms “alkyl” and “alkylenyl” refer to linear or branched aliphatic chains, preferably from 1 to 4 carbon atoms and the term halide to fluorine, bromine or iodine. These compounds are neutral, lipophilic and have low molecular weight and therefore they cross the blood brain barrier and attach to the amyloid sheets. The present invention provides procedures for obtaining naphthalene derivatives with good yields, which can be practical, economical and adapted to a larger-scale manufacturing. We are unaware whether the compounds presented in this invention have been previously reported.

PRIOR RELATED APPLICATIONS

This application is a 371 U.S. National Phase Patent Application andclaims priority to PCT Patent Application No. PCT/CU2009/000057, filedApr. 17, 2009; PCT Patent Application No. PCT/CU2010/000001, filed Apr.17, 2010 and incorporates the above-referenced applications in theirentireties by reference thereto.

DESCRIPTION OF THE INVENTION

The present invention is related to the branch of chemistry,particularly with obtaining new naphthalene derivatives and its labelingwith radionuclides used in nuclear medicine for imaging diagnostic,which includes gamma ray emitters such as technetium 99 metastable(^(99m)Tc), iodine 123 or 131 (¹²³I or ¹³¹I) and others, as well aspositron emitters: fluorine 18 (¹⁸F), carbon 11 (¹¹C), oxygen 15 (¹⁵O)and others, with the aim of displaying neuropathological deposits in thebrain through images from nuclear medicine and providing early diagnosisof Alzheimer's disease or other diseases that might be associated withthe presence of these neuro-deposits (such as rheumatoid arthritis andothers).

Alzheimer's disease (AD) affects about 20 to 40% of the elderlypopulation. It is estimated that, at a global scale, 18 to 22 millionpeople suffer from AD or a related dementia, and it will reach 34million by 2020. This condition is characterized by the presence ofneuropathology deposits in the brain (senile sheets and neurofibrillarytangles), which are involved in the process leading to progressiveneuronal degeneration and neuronal death. The neurofibrillary tangles(NFT) are fibrillar aggregates of hyperphosphorylated tau protein andare located within neuronal cells. In contrast, senile sheets are foundoutside them and consist primarily of deposits of β-amyloid peptides(39-42 amino acids). Gong et al., Proc. Natl. Acad. Sci. USA 2003, 10(18), 10417-22.

The clinical diagnosis of AD has a moderate reliability and often lackssensitivity and specificity. Ball et al., in Neurobiol. Aging. 1997, 18(4), S1-2, suggests that its definitive diagnosis is made post-mortemthrough neuropathological examination with the discovery of senilesheets and/or neurofibrillar tangles in neocortical brain sections,which are displayed through staining (colorimetric or fluorescent) withCongo Red (CR), thioflavin or Chrysamine-G.

Since the histopathologic appearance of these structures occurs longbefore the disease symptoms appear, it is valuable to count with a meansof early and in vivo diagnosis that allows visualizing the same and thatalso facilitates monitoring the effectiveness of the therapeutictreatments.

For this purpose, non-invasive methods made through genetic tests,immunoassays and imageneology techniques are under study. In particular,the latter are very favourable, for both, the AD diagnosis and otherneurological diseases linked to the presence of neurofibrillary tanglesand amyloid sheets, such as: Parkinson's, Down syndrome, hereditarycerebral hemorrhage associated with amyloidosis Dutch type and otherdiseases associated with amyloidosis.

Neuroimaging techniques include: Positron Emission Tomography (PET),Single Photon Emission Computer Tomography (SPECT) and MagneticResonance Imaging (MRI). Volder et al., Developmental Science, 2002, 5(3), 344-60.

To implement the PET technique, it has used different markers utilizedin the in vitro detection of amyloid structures, such as Congo Red,thioflavin and Chrysamine-G analogs, but with unsatisfactory results.Zhen et al., J. Med Chem, 1999, 42, 309-24; Dezutter et al., Eur. J.Nucl. Med, 1999, 26, 1392-99.

Other compounds tested, such as 1-(6 [2-[¹⁸F]fluorethyl)(methyl)amino]naphthalene-2-yl)ethanone (¹⁸F-FENE) and2-(1-(6-[(2-[¹⁸F] fluorethyl)(methyl)amino]-2-naphthyl)ethyl)malononitrile (¹⁸F-FDDNP, Barrio et al. in WO0010614, WO2005040337 andUS2004072371; Kepe et al. in WO2006083378 and Agdeppa at al., 2001, J.Neurosci., 21, 24, 189) labelled with the isotope fluorine 18 (t ½=109.8min), which have been used in the in vivo detection of pathologicaldeposits of AD and detected with the PET technique. It has also beendescribed by Small at al. N. Engl. J. Med 2006, 355, 25, 2652-63, thatit is possible to differentiate patients with cognitive impairment fromthose with AD.

Other compounds have also labeled with fluorine 18 or carbon 11 (t½=20.4 min), which are derived from benzothiazole, imidazole, stilbene,acridine or styrylbenzoxazol with few promising results. The use of PETvisualization technique is limited by: the high cost of equipment, typeof isotope used are generated in a cyclotron located within the nuclearmedicine service or close to it and the short disintegration period,which requires that the labelled radiopharmaceuticals with them must beused immediately. Moreover, imaging SPECT technique is more advantageoussince its equipment is less expensive and the generation and prices ofthe isotopes are more affordable, allowing this technique to be morewidespread.

In particular, technetium 99 m (t_(1/2)=6.02 h., Eγ=140 keV) is used inmore than 80% of routine nuclear medicine diagnosis because it can beused in low doses and therefore the patient receives less radiation.Furthermore, it is obtained from a generator of ⁹⁹Mo/^(99m)Tc, which isavailable on the market at relatively cheap prices. This radioisotopehas as special feature that is able to form stable complexes withorganic compounds related to biological structures, presenting donoratoms. All these advantages point to the need of including a diagnosis,which may be used in SPECT technique, which is reliable forquantification and visualization of amyloid deposits in the brain.

There are numerous patents relating to the use of ^(99m)Tc as a markerto diagnose various brain pathologies.

Among them is the invention of Li-Han et al. (TW438596) which describesthe production of Tropane radiopharmaceutical ^(99m)Tc-TRODAT asselective marker of the dopamine transporter, for the detection ofpresynaptic neuronal degeneration in Parkinson's disease, without rulingout other diseases such as AD. Furthermore, Zhu et al. in CN1072020describes a complex of ^(99m)Tc-ethylcysteinate diethyl ester (ECD),which crosses the blood brain barrier and it is used for cerebralperfusion studies. With this aim, they have studied other neutral andlipophilic complexes of ^(99m)Tc-containing functional groups of amide,amine, thioether, thiol and oximes especially of the propylamineoximinetype (EP0194843, GB8426845D0, EP0123504, EP0229718 and U.S. Pat. No.5,690,904). In general, these patents describe procedures for obtainingsuitable ligands for the ^(99m)Tc, in order to improve retention of thecomplex in the brain, and their in vivo stabilities and guarantee a goodSPECT image quality. However, using these complexes of ^(99m)Tc for thediagnosis of AD has had unsatisfactory results.

Specifically, for the early diagnosis of AD, the aforementioned pigmentshave been marked with ^(99m)Tc, ¹²³I or ¹¹C, used for amyloid structurespostmortem staining, such as the Congo Red and its benzothiazoles andbenzidine derivatives (U.S. Pat. No. 5,008,099, U.S. Pat. No. 6,114,175,U.S. Pat. No. 6,133,259, U.S. Pat. No. 6,417,178). Theseradiopharmaceuticals have shown favourable results in studies in vitro,but failed in vivo diagnosis. In addition, Klunk et al. in U.S. Pat. No.6,168,776 argue that many of these compounds have carcinogenicproperties. Other Congo Red complexes with ^(99m)Tc labeled aredescribed in U.S. Pat. No. 6,379,650 by Wesley et al., where theyobtained neutral and lipophilic ligands using diamine dithiolates.However, these complexes do not cross the blood-brain barrier due to thepresence of sulphonic groups and their high toxicity.

Sharma et al. US2006039859 describes a variation in the use of CongoRed, which is used to label peptides. These new derivatives are capableof crossing the blood brain barrier and bind to the amyloid structures.As an extension of the patent, it is reported that the peptide portionof these derivative functions as a ligand of ^(99m)Tc. However, theresults described are unsatisfactory which may be due to the highmolecular weight of the complexes in question.

Other types of molecules that bind specifically to the deposition ofinsoluble amyloid protein, are styrylbencenes derivatives (Zhuang etal., J. Med. Chem., 2001, 44, 12, 1905-14) and pyridine (Kung et al.,Mol. Imaging. Biol, 2003, 5, 6, 418-26). In particular, derivatives ofstilbene showed by Kung et al. in WO03018070 and WO2006066104 have beeneffective as inhibitors of amyloid aggregation. However, as described bythese authors in Nucl. Med. Biol, 2005, 32, 2, 171-84, conjugates ofthese compounds with ^(99m)Tc have not shown favourable results for thein vivo detection of AD.

It is known, following epidemiological studies, that the use ofnon-steroidal anti-inflammatory drugs reduce the relative risk ofdeveloping AD, among them is naproxen, which count with a naphthalenering (Agdeppa et al., 2003 Neurosciences, 117, 723-30).

On this basis, naphthalene rings present in the Congo Red have beensusceptible to chemical modifications, for the purpose of evaluatingthese compounds for in vivo diagnosis of AD. So, Steven et al. in U.S.Pat. No. 4,933,156 show, among others, the first derivative of Congo Redidentified for this purpose, marked with radioactive isotopes of iodine.Moreover, Kung et al. described in US2006051293 the thioflavinderivatives which may have substituent groups such as naphthyl, amongothers, and which are able to form lipophilic neutral complexes with^(99m)Tc. Also Gallo et al. refer in WO0200603 the use of pamoic acid,its derivatives and analogues, for the treatment of diseasescharacterized by deposition of amyloid aggregates. In particular, pamoicacid is a derivative of naphthoic acid, which has in its structure tworings of naphthalene and forms complexes with radioactive isotopes suchas indium, gadolinium and technetium. In short, in these patentscompounds described are obtained through complex and laborious synthesisprocedures, from expensive raw materials.

Minetti at al. in WO2007045593 describes other naphthyl derivatives,which also inhibit amyloid aggregation and, according to its inventors,surprisingly, cross the blood brain barrier. These compounds, besidesbeing present in pharmaceutical compositions for treating thiscondition, can also be used for diagnosis through different imagingtechniques. In this case, one element of the compounds structure isreplaced by a carbon, hydrogen or oxygen radioactive isotope, or theyalso form stable compounds with radioisotopes of iodine, indium,gadolinium or technetium.

In the application WO02075316 of Wischik et al. is presented a methodfor determining neurofibrillary degeneration associated to tautopatia,as manifested in AD, which describes new ligands of sulphonatedbenzothiazol type. This invention claimed ligands with groups that formcomplexes with technetium and also one of the proposed formulas hasnaphthyl as a substituent group, among others. Similarly, Hays et al. inWO9716194 describes some naphthyl-azo compounds, which inhibit amyloidaggregation and can be labeled with radioisotopes to diagnose. However,there is no data or examples of in vivo experiments that support thisapplication and there is no reference in the claims to a particularradioisotope.

The present invention relates to derivatives of naphthalene, which showhydrophobic properties and are therefore able to cross the blood brainbarrier and are related to characteristic pathological biostructures ofAD. In addition, these compounds have the function of forming stablecompounds with gamma ray emitters such as technetium 99 metastable(^(99m)Tc), iodine 123 or 131 (¹²³I or ¹³¹I) and others, as well aspositron emitters: fluorine-18 (¹⁸F), carbon 11 (¹¹C) and oxygen 15(¹⁵O) and others. Also, these compounds may bear appropriate functionalgroups, such as fluorine-19 atom, which allow the obtaining of nuclearmagnetic resonance imaging. The visualization of these markedbiostructures is done with the proper instrumentation for each case,which allows observing and quantifying the distribution of the labeledcompound within the brain. An extension of this invention's object isthat it provides synthesis procedures of a series of hydrophobiccompounds labeled with gamma emitters or fluorine 19 (¹⁹F). Anotherobject of this invention is that these compounds can be used asdiagnosis of diseases characterized by the appearance of amyloid tissue.They can be used as therapeutic agents of the aforementioned diseases.

This invention provides novel derivatives of naphthalene and itsobtaining procedures through chemical synthesis. In particular, thesenew compounds are characterized by crossing the blood brain barrier andbind selectively to the senile sheets that appear in Alzheimer'sdisease. The present invention involves obtaining derivatives thatcorrespond to the structures I and II.

In structures I and II the R terms are independent.

Where in I: R₁: is selected from the groupalkylenyl-C(O)NH-alkylenyl-R₃, -alkylenyl-C(O)O—R₄. R₃: is selected fromthe group —COOH, —OH, —SH, —NH₂, -alkyl-NH, -alkyl-N-dithiocarbamatealkaline earth metal salts. R₄: is selected from the H group,succinimidyl group, R₂: is selected from the group —H, -alkyl.

II where: R₁: is selected from the group -alkyl, -alkylenyl-halide,-alkylenyl-hydroxyl, -alkylenyl-O-arylsulfonate,alkylenyl-O-alkylsulfonate; R₂: is selected from the group -halide,-alkylenyl-O-arylsulfonate, -alkylenyl-O-alkylsulfonate,-alkylenyl-halide, —CH(O), —HC═C(CN)₂, —HC═CHNO₂, -alkylenyl-NH₂,-alkylenyl-NH-alkyl, -alkyl-alkylenyl-N-dithiocarbamate salts such ascesium, potassium or sodium.

In both structures, the term “alkyl” refers to a straight or branchedaliphatic chain, of saturated carbon and hydrogen atoms, preferablymethyl, ethyl, n-propyl, iso-propyl, n-butyl or iso-butyl. The“alkylenyl” term refers to a divalent analog of a linear or branchedalkyl group, preferably ethylenyl (—CH₂CH₂—), propylenyl (—CH₂CH₂CH₂—)or butylenyl (—CH₂CH₂CH₂CH₂—). The term “halide” refers to fluorine,bromine or iodine.

The present invention also includes tautomeric forms, geometric andoptically active isomers and enantiomers, diastereomers and racemicmixtures of compounds defined by the structures I and II.

This invention has, as one of its objectives, to provide synthesisgeneral procedures for obtaining new derivatives of naphthalene withgood yields, as it is illustrated in FIGS. 1 and 2, which should not beregarded in any way as a constrain of the present invention. Theprocedures are practical, inexpensive and can be adapted tomanufacturing at a larger scale.

In general, FIG. 1 shows the compounds with structure I, which can beobtained from the naphthylamine raw material very accessible in themarket. In principle, the naphthylamine reacts with succinic anhydridein the presence or absence of a tertiary amine (whose pKa is between 4and 8, preferably N-methylmorpholine), to form compounds1-(1-naphthyl)-2,5-pyrrolidinone (1, step a) or4-(1-naphthylamine)-4-oxobutanoic acid (2, step b), respectively.Obtaining these compounds allows introducing, through various reactions,spacer arms in position α-(or 1-) of naphthalene molecule. With the samepurpose, it is also obtained the 4-(1-naphthylamino)-4-oxobutanoic acid,N-hydroxysuccinimide ester (3, step d). The condensation reaction of 2with N-hydroxysuccinimide (NHS) occurs in presence of a condensing agentlike dicyclohexylcarbodiimide (DCC) and preferably with anhydrous1,4-dioxane as solvent. In this synthesis is obtained a high yieldwithout the need of purifying product 3 for later use.

Then, the naphthyl derivative 3 is used as acylating agent. The compound3 is selective for primary amino groups, such as linear or branchedaliphatic diamines (ethylenediamine, propylenediamine,2-ethylenediamine, butylenediamine) and polyfunctional amines such asα-amino acids; thioamines, amino alcohols and aminocarboxylic acids(α-aminoisobutyric acid, 2-aminoethanethiol, ethanolamine, β-alanine,6-aminohexanoico acid, etc.). The reaction conditions are mild(temperature, solvent, time, etc.) and generate no corrosive waste.Finally, with this reaction a further lengthening of the carbon chain ofspacer arm is achieved so as to obtain the following new compounds: 4, 5(step e), 6 and 7 (step c).

These compounds can also be obtained by a new procedure through aone-pot technique, which is an innovation. With this purpose thecompound 3 is obtain in situ, then it reacted with an excess of diamine(e.g. ethylenediamine and butylenediamine) or a aminocarboxylic acid(e.g. β-alanine and 6-aminohexanoic acid) to obtain derivatives 4, 5, 6and 7, which carry an acid or amino terminal group. Specifically, theexcess of the diamine is to ensure that no side products are formed suchas diamines N,N′-disubstituted, leading to a dramatic decrease in thesynthesis yield. The reaction is carried out at room temperature and inthe presence of an organic solvent, preferably anhydrous 1,4-dioxane, toobtain good yields.

In the same way and with similar yields, derivatives 6 and 7 areobtained from the reaction of the naphthyl derivative (1) with thediamine excess (step c*, FIG. 1) at reflux and in the presence of anorganic solvent, anhydrous preferably 1,4-dioxane or DMF. So far, wehave not found reports of these compounds in literature.

This patent also shows the procedure for obtaining derivatives 8 (stepf, FIG. 1), from the reaction of N-alkylation (step f-a) of thepreviously described amino-terminal derivatives (6 and 7), in thepresence of different alkylating agents (CH₃I, C₂H₅Br, (CH₃)₂SO₄,(C₂H₅)₂SO₄), inorganic bases (K₂CO₃, Cs₂CO₃, CsOH) and solvents(acetone, DMF). These new alkyl derivatives (described example: compound8a) react with CS₂ in the presence of CsOH, producing dithiocarbamatecompounds (stage f-b). They can be used as potential ligands of the^(99m)Tc, like its synthetic precursors the alkylamino derivatives.

The iodine labeling methods are classified into direct or indirect,according to the type of bond established between the iodine and thecompound to be marked. In direct methods, radioactive iodine is easyincorporated with high efficiency to the aromatic ring of organiccompounds. In this invention this procedure is used to label thecompound 6 (step g, FIG. 1), specifically through the Iodogeno orchloramide method (Saha on Fundamentals of Nuclear Pharmacy.Radiopharmaceutical and Methods of Radiolabeling. Fourth EdSpringer-Verlag, USA. 1998, p.: 93-97). This procedure has a labelingefficiency ranging between 70 and 80% and high specific activity sincethere is no isotope or sample dilution. This labeling procedure can beextended to other molecules described here that have another spacer arm.We are not aware of literature reports regarding these labeledcompounds.

On the other hand, the compounds with structure II can be obtained fromthe affordable 2-naphthol raw material. The general procedure shown inFIG. 2, which consists of 5 stages of synthesis, specifically aims tointroduce a mono-fluorinated alkyl chain in position 6- of the2-methoxynaphthalene.

The first stage is the bromination of 2-naphthol (pKa 9.23) carried outthrough a procedure already described by Koelsch in Organic Synthesis,Coll., 1955, 3, 132 and Reddy et al., in Organic process research andDevelopment, 1999, 3, 121-25. Thus, the 2-naphthol reacts with molecularbromine in presence of glacial acetic acid as solvent and Sn as reducingagent of the 1,6-dibromo-2-naphthol intermediate, which is in situformed. This reduction is selective due to the dibrominated derivativethermodynamic instability leading to 6-bromo-2-naphthol (10).

The step b of FIG. 2 is the reaction of O-alkylation of 10 to obtain the6-bromo-2-methoxynaphthalene (11). Reddy et al. described this procedurein Organic Process Research and Development, 1999, 3, 121-25, from thereaction between 10 and dimethylsulfate as alkylating agent in thepresence of K₂CO₃ as base and a mixture of acetone-water as solvent, at60° C. However, under these conditions the degree of conversion is verylow, even though DMF or acetone are used as solvents and the reaction isrefluxed for 3 to 72 hours. The use of cesium bases (CsOH and Cs₂CO₃)compared to their counterparts in Li⁺, Na⁺, K⁺ and Rb⁺, is currentlyreported in the alkylation reactions due to the so-called “cesiumeffect”. (Welton in Chem. Rev., 1999, 99, 2071-83; Kim et al. in J. Am.Chem. Soc. 2002, 124, 10278-9; Gerstenberger et al. in Angew. Chem. Int.Ed. Engl., 1981, 20, 647-67). In this invention, Cs₂CO₃ base aresuccessfully used in the reaction of O-alkylation of 10 with differentalkylating agents (alkyl sulfates, alkyl halides, alkyl dihalides). Thereaction occurs rapidly with dimethylsulfate in acetone (as solvent) atroom temperature. The yield of compound 11 is practically quantitative.We are not aware that these compounds of cesium, in particular Cs₂CO₃,are used for the reaction conditions described.

In the third stage of synthesis (step c, FIG. 2) 11 reacts with Mg inthe presence of iodine traces to obtain the Grignard reagent (12),according to general procedure described by Kidwell et al. (OrganicSynthesis, Coll., 1973, 5, 918). This intermediate is not isolated andit is subsequently used in cross-coupling reaction (step d). In thepresent invention, this synthesis is performed, in general, from areaction of an aryl magnesium with a halide or an alkyl sultanate and acopper complex as catalyst. In this procedure, carried out in an inertatmosphere, the order of the reagent addition and temperature play animportant role. Specifically, if it is added the Grignard reagent 12 tothe mixture of 1,3-propanediol di-p-tosylate with the copper catalytic(Li₂CuCl₄ or Li₂CuCl₃) which is between −70° C. to −15° C.,3-(6-methoxy-2-naphthyl)propyl 4-methylbenzenesulfonate (13) is obtainedwith good yields. This new compound, 13, is a valuable intermediate inthe next synthetic stage which is an innovation of this patent. Thefourth stage of synthesis (step e, FIG. 2) refers to the obtaining of afluorinated derivative, based on studies by Kim et al. in J. Org. Chem.,2003, 68, 4281-5. This nucleophilic substitution reaction occurs withthe use of an alkali metal fluoride (KF, CsF and RbF) in the presence ofan ionic liquid, such as 1-n-butyl-3-methylimidazolium (bmim) (BF₄ ⁻,PF₆ ⁻, SbF₆ ⁻, triflate [OTf], bis(trifluoromethanesulfonyl)imide [NTf₂⁻], OAc⁻) and acetonitrile as solvent. In this invention, the reactiontakes place with 13 preferably, in the presence of CsF and (bmim) (BF₄⁻) to obtain the new derivative 2-(3-fluoropropyl)-6-methoxynaphthalene(14).

Chemical compounds that cross the blood brain barrier (BBB) should beneutral and lipophilic and also, have a low molecular weight. Thepartition coefficient (P) provides a measure of the compoundlipophilicity and is closely related to drug distribution in the body,its absorption in tissues and its route of excretion (Meade et al. inCurr. Opin. Neurobiol., 2003, 13, 5, 597-602). One method used todetermine this parameter is that of radiotracers which relates theradiotracers activity or number of counts in the octanol organic phasewith that of the aqueous phase. In this invention the compounds labelingis carried out using the Iodogeno method (¹³¹I).

According to studies Dischino et al. (The Journal of Nuclear Medicine,1983, 24, 11, 1030-38), the optimal values of partition coefficient,which ensure that the compound crosses the BBB, are in the range of 0.9to 2.5. On the other hand, there has been established by J. Levin inMed. Chem., 1980, 23, 682-84, that to cross the BBB by passivediffusion, the molecular weight values must range between 400 and 657Da. In this invention, the synthesized compounds have values ofmolecular weights between 200 and 450 Da, and their partitioncoefficients, expressed as log P measured in octanol/water, rangebetween 2.1 and 2.5.

The current invention shows that the synthesized compounds have valuesof molecular weights and partition coefficients that correspond to thevalues set for other compounds that cross the BBB. In summary, thecompounds described herein, have the advantage of being used as stainingagents of amyloid plaques present in AD as they show correspondence withthese parameters.

In this invention the compositions of naphthalene derivatives labeledwith ¹³¹I, injected into rats, rapidly cross the blood brain barrier andhave an appropriate retention time.

As non-limiting example, FIG. 3 shows the distribution in rats ofcompound 6 labeled with ¹³¹I, (9) after its intravenous injection. Thus,an arrow indicates the detection of the compound in the region ofinterest (maximum uptake at about 3 min.) and then starts the slowexcretion of the radioiodinated compound.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the general procedure for synthesis of derivatives1-naphthyl derivatives with structure I, which includes the mostrelevant reaction conditions (a: succinic anhydride, NMM; b: succinicanhydride, c, c*: ethylenediamine or 1,4-butylenediamine d: NHS, DCC, e:6-aminocaproic acid or β-alanine, f: a) CH₃I, base, b) CS₂, CsOH; g:Iodogeno in CHCl₃ (¹³¹I).

FIG. 2 shows the general procedure of synthesis of 2,6-naphthylderivatives with structure II, which includes the most significantreaction conditions (a: Br₂, Sn, glacial acetic acid; b:dimethylsulfate, base, acetone; c,: Mg, THF, Ar, d: 1,3-propanedioldi-p-tosylate (PrDiTs) Li₂CuCl₄, THF, Ar; e: 1-butyl-3-methylimidazoliumtetrafluoroborate, CsF, CH₃CN).

FIG. 3 shows the distribution of the N1-(2-aminoethyl)-N4-(1-naphthyl)succinimide (6) compound labeled with ¹³¹I in rats, after itsintravenous injection. The arrow indicates the detection of the compoundin the region of interest (maximum uptake at about 3 min.).

The obtaining procedures of naphthalene derivatives showed in thepresent invention are further illustrated by the following examples,which should not be regarded in any way, as constrain of the presentinvention. The compounds obtained were also appropriately characterizedby spectroscopic techniques such as IR, ¹H and ¹³C NMR and Mass.

DESCRIPTION OF THE EMBODIMENTS EXAMPLE 11-(1-naphthyl)-2,5-pyrrolidinedione (1)

1-naphthylamine (5 g, 34.96 mmol) was dissolved in 50 mL of anhydrous1,4-dioxane. Succinic anhydride (6.99 g, 69.92 mmol) andN-methylmorpholine (NMM, 7.6 mL, d=0.92 g/mL, 69.92 mmol) were added.The reaction mixture was heated at reflux for 5 hours. Once the reactionis completed (TLC), the solvent is removed by rotoevaporation. The solidresidue is re-dissolved and re-crystallized from ethanol to obtain 5.2 gof compound 1 (Yield: 66%). Mp.: 153.5-154.5° C. (Lit: 153° C.). ESI-MS(m/z)=226 (M+1)⁺.

EXAMPLE 2 4-(1-naphthylamino)-4-oxobutanoic acid (2)

1-naphthylamine (10 g, 69.84 mmol) and succinic anhydride (13.96 g, 140mmol) were dissolved in 100 mL of anhydrous 1,4-dioxane. The reactionmixture was refluxed for 2 h and later, cooled to precipitate a violetsolid which was filtered and washed with 1,4-dioxane. Recrystallizationfrom ethanol gave 15.82 g of white solid. Yield: 93%. Mp.: 167-169° C.ESI-MS (m/z)=244 (M.+1)⁺.

EXAMPLE 3 4-(1-naphthylamino)-4-oxobutanoic acid, N-hydroxysuccinimideester (3)

Compound 2 (2 g, 8.23 mmol), N-hydroxysuccinimide (NHS) (1.42 g, 12.34mmol) and dicyclohexylcarbodiimide (DCC) (2.54 g, 12.33 mmol) weredissolved in 32 mL of anhydrous 1,4-dioxane. The reaction mixture wasrefluxed for 3 h. and then cooled at room temperature in order to removethe dicyclohexylurea (DCU) formed. The filtrate was rotoevaporated andcooled to separate by filtration a white solid that was washed withplenty of water and diethyl ether, and air-dried to obtain 2.29 g ofproduct. Yield: 87%, Mp.: 168-171° C. ESI-MS (m/z)=341 (M.+1)⁺.

EXAMPLE 4 N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine (4)

Compound 2 (500 mg, 2.06 mmol) was dissolved in 13 mL of anhydrous1,4-dioxane, and to this solution was added NHS (283 mg, 2.46 mmol) andDCC (507 mg, 2.46 mmol). The reaction mixture was refluxed for 3 h. andthen cooled at room temperature in order to remove the dicyclohexylurea(DCU) formed. Then, β-alanine (248 mg, 2.78 mmol was added and themixture was heated for 20 h. at 60° C. The solution was rotoevaporatedto dryness and the crude product was purified by column chromatographywith chloroform as mobile phase. 230 mg of a white solid was obtained.Yield: 40%. Mp.: 180.5-181.8° C. ESI-MS (m/z)=316 (M.+1)⁺.

EXAMPLE 5 6-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}hexanoic acid (5)

2 (1 g, 4.4 mmol), DCC (0.9 g, 4.4 mmol), 6-aminocaproic acid (0.6 g,7.6 mmol) and triethylamine (0.6 mL, 4.3 mmol) in 40 mL of DMF wererefluxed for 6 hours. Then, the mixture reaction was rotoevaporated todryness and the crude product was recrystallized from ethanol. The solidthus obtained was dried over P₂O₅ to yield 0.28 g of 5. Yield: 18%. Mp.:229-231° C. ESI-MS (m/z)=344 (M.+1)⁺.

EXAMPLE 6 N1-(2-aminoethyl)-N4-(1-naphthyl) succinimide (6)

Method A (One Pot): 2 (500 mg, 2.06 mmol) and NHS (283 mg, 2.46 mmol)were dissolved in 5 mL of anhydrous under dry N₂ atmosphere. Next, asolution of DCC (507 mg, 2.46 mmol) in 8 mL of anhydrous 1,4-dioxanewere added dropwise through a pressure equalizing dropping funnel. Thereaction mixture was refluxed for 2 h. and then cooled at roomtemperature in order to remove the dicyclohexylurea (DCU) formed. Asolution of ethylenediamine (0.18 mL, 2.7 mmol) in 1 mL of 1,4-dioxanewas added. The reaction mixture was stirred at room temperature for 30min. and then cooled to precipitate a white solid that was filtered,washed with 1,4-dioxane and acetone, and air-dried to yield 584 mg ofthe product (98%). Recrystallization from ether yielded 85% of pure 6.Mp.: 128.9-130.5° C.

Method B: Compound 1 (2.58 g, 11.47 mmol) and ethylenediamine (7.66 mL,11.4 mmol) in 30 ml of 1,4-dioxane were refluxed for 2 h. and thencooled at room temperature. 15 mL of diethyl ether was added toprecipitate a white solid that was filtered and washed with diethylether to obtain 3.27 g of product. Recrystallization from acetoneyielded 63% of pure 6. Mp.: 128.9-130.5° C. ESI-MS (m/z)=287 (M.+1)⁺.

EXAMPLE 7 N1-(4-aminobutyl)-N4-(1-naphthyl) succinamic (7)

Method A: Compound 2 (2.12 g, 8.72 mmol), NHS (1.42 g, 12.34 mmol) andDCC (2.54 g, 12.33 mmol) in 30 mL of anhydrous 1,4-dioxane were refluxedfor 3 h. and then cooled at room temperature to remove the DCU formed.Butylenediamine (2.6 mL (26.14 mmol) was slowly dropped and the mixturereaction was stirred for 30 min. at room temperature. The mixture wasrotoevaporated to dryness and the crude product re-dissolved in 10 ml ofCHCl₃ was washed with water (3×5 mL). Next, the organic phase was driedwith anhydrous Na₂SO₄ and rotoevaporated to dryness. Diethyl ether (10mL) was added to precipitate in cold a white solid. Yield: 72%. Mp.:142.2-150.8° C.

Method B

Compound 1 (1.832 g, 8.1 mmol), triethylamine (1.94 mL, 14 mmol) and1,4-butylenediamine (1.66 mL, 16.6 mmol) in 30 mL of DMF were refluxedfor 1 hour. After solvent elimination, the reaction mixture was purifiedby column chromatography with ethyl acetate and ethyl acetate:methanol(10:2), as mobile phases. Yield: 11%. Mp.: 142.2-150.8° C. ESI-MS(m/z)=315 (M.+1)⁺.

EXAMPLE 8 N1-[2-(methylamino)ethyl]-N4-(1-naphthyl) succinimide (8a)

A slurry of activated molecular sieves 4 Å (500 mg) and CsOH.H₂O (280mg, 1.7 mmol) in 8 mL of anhydrous DMF, was stirred for 10 minutes.Next, compound 6 (485 mg, 1.7 mmol) in 1 mL of anhydrous DMF was addedto maintain stirring for 30 min. at room temperature. To this reactionmixture, CH₃I (124 μL, 2 mmol) in 0.5 mL of anhydrous DMF was added andstirred for 24 h., at room temperature. The mixture was thus filteredand rotoevaporated to dryness. The crude product was washed with NaOH(1N) and extracted with ethyl acetate. The organic phase was dried withNa₂SO₄ and purified by column chromatography with ethyl acetate asmobile phase. Yield: 70%. Mp.: 120° C. (dec.). ESI-MS (m/z)=300 (M.+1)⁺.

EXAMPLE 9 Sodium Salt of the Acid Methyl(2-([4-(1-naphthylamine)-4-oxobutanoil]amino)ethyl)carbamoditionic (8b)

Compound 8a (299 mg, 1 mmol) was added to a suspension of NaOH (80 mg, 2mmol) in 3 mL of dry diethyl ether. The reaction mixture was cooled inan ice bath and stirred vigorously for 30 min., to slowly drop CS₂ (121μL, 2 mmol) in 0.5 mL of ether. Then, the mixture was stirred for 30min. in cold and then, at room temperature for 2 hours. Solids werefiltered and washed with dry diethyl ether. Yield: 75%. Mp.: (dec.).ESI-MS (m/z)=398 (M.+1)⁺.

EXAMPLE 10 N1-(2-aminoethyl)-N4-(1-naphthyl) succinimide-¹³¹I (9)

Labeling with ¹³¹I: To a tube with Iodogeno covered walls (the tubeswere impregnated with 250 to 500 μL of a Iodogeno solution (0.2 mg/mL inCHCl₃), under in dry nitrogen atmosphere) was added 503.2 MBq (13.6 mCi)of ¹³¹I and stirred for 10 min. at room temperature. Then 100 μL of asolution of 6 (7.7 10⁻³ mol/L) in PBS (pH 8.5) was added and thereaction mixture was thus stirred for another 15 minutes. The mixturewas decanted in order to remove the ¹³¹I free by filtration throughfilters of 3 MM Whatman paper impregnated with silver nitrate.

EXAMPLE 11 6-bromo-2-methoxynaphthalene (11)

6-Bromo-2-naphthol (10) was obtained from the reaction between2-naphthol and molecular bromine in glacial acetic acid according toprocedure described by Reddy et al. in Organic Process Research andDevelopment, 1999, 3, 121-25.

To a solution of Cs₂CO₃ (7.55 g, 23.3 mmol) and 6-bromo-2-naphthol (4.6g, 17.9 mmol) in 45 mL of acetone, (CH₃)₂SO₄ (2.2 mL, 23.3 mmol) wasadded dropwise and stirred for 30 min. at room temperature. The reactionmixture was rotoevaporated and the crude was washed with water andextracted with CHCl₃. The organic phase was dried with MgSO₄ and cooledto precipitate 4.45 g of a white solid. Yield: 91%. Mp.: 101.4-103.7° C.(lit.: 103-105° C.).

EXAMPLE 12 3-(6-methoxy-2-naphthyl)propyl 4-methylbenzenesulfonate (13)

Preparation of Grignard Reagent: According to general proceduredescribed by Kidwell et al. in Organic Synthesis, Coll., 1973, 5, 918.In particular, in this invention, the reagent obtaining process wascarried out in a flask with Mg (0.363 g, 14.95 mmol) and a smallcrystals of I₂, previously flame-dried and the atmosphere replaced withdry Ar. Then, 1 mL (0.709 g, 2.99 mmol) of a solution of 11, in 3 mL ofTHF, was added dropwise to the flask. The reaction mixture was slowlyheated to reflux until the boiling becomes spontaneous and a whitesludge was formed. After that, the rest of the solution was addeddropwise and refluxed for 4 h. until the formation of 12.

Preparation of the Catalyst Li₂CuCl₄:

According to general procedure described by Burns et al., in J. Chem.Soc. 1997, 119, 2125-2133.

To a flask, previously flame-dried and evacuated with Ar, a solution of1,3-propanediol di-p-tosylate (PrDiTs) (1.26 g, 3.289 mmol) in 1 mL ofTHF was added. Then, 1.79 mL of catalyst Li₂CuCl₄ was added. Thereaction mixture was cooled at −30° C. and the Grignard reagent wasadded dropwise. Once the addition process was finished, the mixture wasmaintained at 8° C. for 24 h. and later at room temperature for 48hours. The product was thus purified by column chromatography with amixture of n-hexane:dichloromethane (100:0 to 80:20) as mobile phase.Yield: 15%. Mp.: dec. ESI-MS (m/z)=371 (M+1)⁺.

EXAMPLE 13 2-(3-fluoropropyl)-6-methoxynaphthalene (14)

3 mL of (bmim)(BF₄) was added to 3 mL of a solution of H₂O (90 μL, 5mmol) in CH₃CN with 13 (370 mg, 1 mmol). Then, anhydrous CsF (760 mg, 5mmol) was added. The reaction mixture was stirred at 100° C. for 2hours. Upon completion of the reaction, the product of interest wasextracted with diethyl ether (3×5 mL). The organic phase was dried(MgSO₄) and concentrated to dryness to purify by column chromatography(ethyl acetate:hexane as mobile phase). Yield: 40%. Mp.: dec. ESI-MS(m/z)=219 (M+1)⁺.

EXAMPLE 14 Labeling Studies and Biodistribution in Rats. Compound:N1-(2-aminoethyl)-N4-(1-naphthyl) succinimide-¹³¹I (9)

Determination of Partition Coefficients: A solution of 9 (20 μL),prepared as described in example 10, was added to a mixture of 3 ml ofn-octanol and 3 ml of distilled water. After stirring and leaving tostand the mixture, aliquots of 20 μL from each phase were taken todetermine the radiometric distribution ratio by an activity meter (CRC35R, Capintec Inc.). This procedure was repeated to theradiopharmaceutical of ^(99m)Tc-ECD as reference, which is used forcerebral perfusion studies. Partition coefficient of 9: 0.54(logP=−0.27) and ^(99m)Tc-ECD: 40.6 (logP=1.6).

Animal Studies: A solution of 9 15 MBq (407 Ci), prepared as describedabove, was administered to male Wistar rats (160 g, n=3) through thelateral tail vein. Then, images were taken every 15 seconds, for 30 min.(120 images) with a gamma camera (Medis Nucline TH22, Hungary), with apeak centered at 360 keV and a window of ±25%. The image processing wasperformed on a processing station Segami (USA).

The invention claimed is:
 1. An amyloid binding compound, said compoundcomprises structure I;

wherein; R₁ is -alkylenyl-C(O)NH-alkylenyl-R₃, or -alkylenyl-C(O)O—R₄,R₃ is —COOH, —OH, —SH, —NH₂, —NH-alkyl or —N(-alkyl)-dithiocarbamatealkaline earth metal salts, R₄ is a succinimidyl group, and R₂ is H oralkyl, and wherein at least one or more of the oxygen or carbon atomsare substituted by a corresponding radioactive isotope or at least oneof the hydrogen atoms of aromatic ring is substituted by halogen;wherein the compound has a partition coefficient LogP value of less than1.6 so as to readily pass the blood brain barrier and selectively bindto amyloid sheets in a mammalian brain; whereby the amyloid bindingcompound is readily displayable in the mammalian brain.
 2. The amyloidbinding compound of claim 1, wherein the alkylenyl in R₁ is selectedfrom the group consisting of ethylene (—CH₂CH₂—) and butylenyl(—CH₂CH₂CH₂CH₂—); and the alkyl in R₂ is selected from the groupconsisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl andiso-butyl.
 3. The amyloid binding compound of claim 1, wherein R₁ is—(CH₂)_(n)CONH(CH₂)_(m)—R₃ (n=2, 3 or 4 and m=2, 3, 4, 5 or 6), R₃ is—COOH; and wherein R₂ is —H or -alkyl.
 4. The amyloid binding compoundof claim 1, wherein R₁ is —(CH₂)_(n)CONH(CH₂)m-R₃ (n=2, 3 or 4 and m=2,3, 4, 5 or 6), R₃ is —NH₂, —NHCH₃ or —NHC₂H₅ and R₂ is —H or -alkyl. 5.The amyloid binding compound of claim 1, wherein R₁ is—(CH₂)_(n)CONH(CH₂)_(m)—R₃(n=2, 3 or 4 and m=2, 3, 4, 5 or 6), R₃ is—NR₅R₆, R₅ is —CH₃ or —C₂H₅, R₆ is C(S)SW, W is Na⁺, K⁺ or Cs⁺ and R₂ is—H or -alkyl.
 6. The amyloid binding compound of claim 1, wherein R₁ is—(CH₂)_(n)CONH(CH₂)m-R₃ (n=2, 3 or 4 and m=2, 3, 4, 5 or 6), R₃ is —OHand R₂ is —H or -alkyl.
 7. The amyloid binding compound of claim 1,wherein R₁ is —(CH₂)_(n)CONH(CH₂)_(m)—R₃ (n=2, 3 or 4 and m=2, 3, 4, 5or 6), R₃ is —SH and R₂ is —H or -alkyl.
 8. The amyloid binding compoundof claim 1, wherein R₁ is —(CH₂)_(n)C(O)O—R₄ (n=2, 3 or 4), R₄ is asuccinimidyl group and R₂ is —H or -alkyl.
 9. The amyloid bindingcompound of claim 1, wherein R₁ is -alkylenyl-C(O)NH-alkylenyl-R₃, andR₃ is selected from the group consisting of —COOH, —OH, —SH, —NH₂,—NH-alkyl and —N(-alkyl) -dithiocarbamate alkaline earth metal salts, inwhich at least one of the hydrogen atoms of aromatic ring is replaced bya radioactive halogen atom selected from the group consisting of ¹²³I,¹³¹I and ¹⁸F.
 10. The amyloid binding compound of claim 1, wherein R₁ is-alkylenyl-C(O)NH-alkylenyl-R₃, and R₃ is selected from the groupconsisting of —COOH, —OH, —SH, —NH₂, —NH-alkyl and —N(-alkyl)-dithiocarbamate alkaline earth metal salts, which is complexed with^(99m)Tc—, and a chelating ligand, wherein the chelating ligand isselected from the group consisting of 2,2′-oxydiethanethiol andN¹-(2-aminoethyl)-1,2-ethanediamine.
 11. The amyloid binding compound ofclaim 1, wherein at least one of the oxygen or carbon atoms aresubstituted by a corresponding radioactive isotope selected from thegroup consisting of ¹¹C and ¹⁵O.
 12. The amyloid binding compound ofclaim 1, wherein R₁ is -alkylenyl-C(O)NH—alkylenyl-R₃, and R₃ isselected from the group consisting of —COON, —OH, —SH, —NH₂, —NH-alkyland —N(-alkyl)-dithiocarbamate alkaline earth metal salts, in which atleast one of the hydrogen atoms of aromatic ring is replaced by afluorine atom.
 13. The amyloid binding compound of claim 9, wherein theamyloid binding compound is readily displayable in the mammalian brainby single photon emission computed tomography (SPECT) or positronemission tomography (PET) techniques.
 14. The amyloid binding compoundof claim 11, wherein the amyloid binding compound is readily displayablein the mammalian brain by positron emission tomography (PET) technique.15. The amyloid binding compound of claim 12, wherein the amyloidbinding compound is readily displayable in the mammalian brain bymagnetic resonance imaging (MRI) technique.
 16. The amyloid bindingcompound of claim 10, wherein the amyloid binding compound is readilydisplayable in the mammalian brain by single photon emission computedtomography (SPECT) technique.